molecules Article
Tomato Aqueous Extract Modulates the Inflammatory Profile of Immune Cells and Endothelial Cells Joseph Schwager *, Nathalie Richard, Bernd Mussler and Daniel Raederstorff DSM Nutritional Products, P. O. Box 2676, Basel 4002, Switzerland;
[email protected] (N.R.);
[email protected] (B.M.);
[email protected] (D.R.) * Correspondence:
[email protected] or
[email protected]; Tel.: +41-61-815-8842 Academic Editor: Norbert Latruffe Received: 9 December 2015; Accepted: 20 January 2016; Published: 29 January 2016
Abstract: Nutrients transiently or chronically modulate functional and biochemical characteristics of cells and tissues both in vivo and in vitro. The influence of tomato aqueous extract (TAE) on the in vitro inflammatory response of activated human peripheral blood leukocytes (PBLs) and macrophages was investigated. Its effect on endothelial dysfunction (ED) was analyzed in human umbilical vein endothelial cells (HUVECs). Murine macrophages (RAW264.7 cells), PBLs and HUVECs were incubated with TAE. They were activated with LPS or TNF-α in order to induce inflammatory processes and ED, respectively. Inflammatory mediators and adhesion molecules were measured by immune assay-based multiplex analysis. Gene expression was quantified by RT-PCR. TAE altered the production of interleukins (IL-1β, IL-6, IL-10, IL-12) and chemokines (CCL2/MCP-1, CCL3/MIP-1α, CCL5/RANTES, CXCL8/IL-8, CXCL10/IP-10) in PBLs. TAE reduced ED-associated expression of adhesion molecules (ICAM-1, VCAM-1) in endothelial cell. In macrophages, the production of nitric oxide, PGE2 , cytokines and ILs (TNF-α, IL-1β, IL-6, IL-12), which reflects chronic inflammatory processes, was reduced. Adenosine was identified as the main bioactive of TAE. Thus, TAE had cell-specific and context-dependent effects. We infer from these in vitro data, that during acute inflammation TAE enhances cellular alertness and therefore the sensing of disturbed immune homeostasis in the vascular-endothelial compartment. Conversely, it blunts inflammatory mediators in macrophages during chronic inflammation. A novel concept of immune regulation by this extract is proposed. Keywords: chronic inflammation; endothelial dysfunction; human umbilical endothelial vein cells; inflammation; innate immune response; macrophages; nutrients; peripheral blood leukocytes
1. Introduction Numerous epidemiological studies established the beneficial relationship between a diet rich in fruit and vegetables and health conditions like cardiovascular diseases (CVD), diabetes, obesity, neuro-degeneration and arthritis. Low-grade inflammation is a common feature of these chronic diseases. This is reflected by an unbalanced production of inflammatory mediators including cytokines and chemokines, a disturbed homeostasis of cellular oxidants and mediators of inflammation such as prostaglandins, nitric oxide or the status of the extracellular matrix (ECM). The homeostatic changes influence cell metabolism and alter tissue functions that might favor disease progression [1]. A hallmark of acute inflammation is the increased production of cytokines and chemokines. These enable and enhance inflammatory processes that are essential to recruit immune cells to the sites of inflammation and eliminate pathogens. Some mediators are further required during the resolution of inflammation [2] or for the differentiation of cells that orchestrate the resolution of inflammatory processes such as alternatively activated macrophages [3]. Chronic inflammation maintains a status of un-coordinated production of mediators and metabolites that cause tissue and organ damage. Molecules 2016, 21, 168; doi:10.3390/molecules21020168
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Inflammatory processes have thus an intrinsic dual nature during acute and chronic inflammation. Therefore, substances that modulate the inflammatory status and maintain immune homeostasis in a context-dependent way are expected to prevent the occurrence of health disorders and diseases [4]. In this study we investigated the in vitro effects of a tomato aqueous extract (TAE) on inflammatory responses. Previous studies with TAE revealed that it improved the blood flow by reducing platelet adhesion and aggregation [5–7]. Likewise, tomato extracts and their lipophilic constituents influenced various mediators of the inflammatory response [8] (reviewed in [9]). In order to cover a potentially wide range of actions in different systemic contexts, we analyzed the effects of TAE in various cellular systems, i.e., human peripheral blood leukocytes (PBLs), human umbilical vein endothelial cells (HUVECs) and macrophage cells (RAW264.7 cells). We found that TAE markedly altered the production of metabolites related to the acute inflammatory response. TAE modulated the transcription factors of the NF-κB signaling pathway and thus regulated expression of inflammatory genes and the production of inflammatory mediators. We show that TAE orchestrates the response of cells to inflammatory stimuli or altered homeostasis in a cell- and compartment-specific way. 2. Results 2.1. Tomato Aqueous Extract Changed the Inflammatory Profile of Murine RAW264.7 Cells Initially, we investigated the influence of TAE on the inflammatory profile of murine macrophages (i.e., RAW267.4 cells) stimulated with E. coli lipopolysaccharide (LPS), which triggered numerous metabolic changes [10]. TAE reduced the LPS-induced production of nitric oxide (NO) and it also significantly diminished the secretion of COX-2 dependent PGE2 (Figure 1). Furthermore, we evaluated the effect of TAE on cytokine and chemokine (CK) production in murine macrophages. TAE concentration-dependently blunted TNF-α and IL-12(p70), while the production of anti-inflammatory IL-10 was augmented (Figure 1). Conversely, TAE had little impact on IL-1β and IL-6. Secretion of chemokines, such as CCL2/MCP-1, CCL4/MIP-1β and CCL5/RANTES, was increased by TAE (Figure 1, Table 1). We further investigated how the expression of inflammatory genes was influenced by TAE. Gene microarray analysis revealed that LPS induced robust up-regulation of hundreds of genes in RAW264.7 cells ([11] and our unpublished results). TAE diminished mRNA levels of TNF-α, IL-6, CCL4/MIP-1β, CCL5/RANTES and CXCL10/IP-10 (Figure 2). The NF-κB transcription pathway was impaired by TAE, as illustrated by reduced expression levels of NF-κB and Iκ-Ba mRNA. This suggests that TAE regulated gene expression via the NF-κB pathway (Supplementary Materials Table S1). Table 1. Effects of constituents of TAE on the secretion of inflammatory metabolites by RAW264.7 macrophages. Cells were stimulated with LPS in the presence of the indicated substances and cultured for 24 h. Metabolites were determined in the culture supernatants by multiplex ELISA and Griess reaction (for nitric oxide). Representative data obtained in one of three different experimental series are shown. Mean values ˘ SD (of triplicate cultures) are given.
Metabolite PGE2 [pg/mL] Nitric oxide (µM) IL-6 [ng/mL] IL-1β [pg/mL] IL-12 [pg/mL] TNF-α [ng/mL] CCL2/MCP-1 [ng/mL] CCL4/MIP-1β [pg/mL] CCL5/RANTES [ng/mL]
LPS Stimulated
TAE (500 µg/mL) +LPS
p
Adenosine (25 µM) +LPS
p
Chlorogenic Acid (25 µM) +LPS
p
Rutin (25 µM) +LPS
p
7034 ˘ 186 18.1 ˘ 0.2 33.6 ˘ 2.4 702 ˘ 120 1083 ˘ 165 293 ˘ 33
5234 ˘ 260 13.1 ˘ 0.8 36.9 ˘ 1.8 579 ˘ 105 1045 ˘ 49 290 ˘ 12
0.09 0.05 0.26 0.39 0.79 0.93
6373 ˘ 44 19.6 ˘ 0.9 24.1 ˘ 2.1 625 ˘ 78 1150 ˘ 127 202 ˘ 5
0.23 0.94 0.05 0.52 0.69 0.07
12,016 ˘ 574 12.2 ˘ 0.1 27.0 ˘ 1.7 567 ˘ 32 1058 ˘ 127 241 ˘ 21
0.05 0.02 0.08 0.26 0.69 0.23
12,940 ˘ 1882 14.4 ˘ 0.2 25.7 ˘ 2.0 498 ˘ 22 921 ˘ 35 292 ˘ 10
0.07 0.04 0.07 0.14 0.31 0.98
9.8 ˘ 1.9
14.9 ˘ 3.7
0.22
6.4 ˘ 2.9
0.30
8.8 ˘ 0.4
0.53
6.7 ˘ 1.7
0.22
792 ˘ 50
1385 ˘ 63
0.01
753 ˘ 31
0.45
698 ˘ 36
0.16
793 ˘ 33
0.99
34.1 ˘ 0.4
58.7 ˘ 1.5
0.002
28.6 ˘ 3.1
0.13
27.7 ˘ 0.8
0.01
28.4 ˘ 2.1
0.06
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3 of 15 LPS only TAE
25
LPS only TAE 3000
PGE2 (pg/mL)
Nitrite (μM)
20 15
*
10
*
2500 2000 1500 1000
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TNF-α
400000 350000
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200000
40000
700
35000
400
100000
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50000
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IL-12(p70)
900 800 700 600 500
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+ FFII 15.6
+ FFII 62.5
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CCL4/MIP-1β
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500
- LPS + FFII + LP FFII+ LP FFII+ LP FFII +LP none LPS - 15.6 62.5 250 1000 TAE (μg/mL) - only 70000
**
CCL5/RANTES
60000 50000
800000
** ** **
40000 30000
600000
6000
200
4000
400000
100
2000
200000
0
0
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- LPS + FFII + LP FFII+ LP FFII+ LP FFII +LP none LPS - 15.6 62.5 250 1000 TAE (μg/mL) - only
300
100
1400000
14000
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200
+ + LPS only FFII LPS 15.6 TAE (μg/mL) -
*
16000
**
FFII+
CCL2/MCP-1
20000
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+ FFII
pg/mL
1000
0+ LPS TAE (μg/mL) -
**
400
20000 15000
IL-10
500
25000
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- LPS + FFII + LP FFII+ LP FFII+ LP FFII +LP none LPS - 15.6 62.5 250 1000 TAE (μg/mL) - only
pg/mL
800
600
pg/mL
250000
pg/mL
pg/mL
300000
IL-6
45000
IL-1β
900
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pg/mL
μg/mL
10
1000
pg/mL
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pg/mL
10 450000
* *
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+ + LPS only FFII LPS 15.6 TAE (μg/mL) -
+ FFII
62.5
+ FFII
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FFII+
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20000 10000 0
+ + LPS only FFII LPS 15.6 TAE (μg/mL) -
+ FFII
62.5
+ FFII
250
FFII+
500
+ + LPS only FFII LPS 15.6 TAE (μg/mL) -
+ FFII
62.5
+ FFII
250
FFII+
500
Figure 1. TAE inhibits the production of NO and PGE2 and modulates cytokine and chemokine Figure 1. TAE inhibits the production of NO and PGE2 and modulates cytokine and chemokine secretion in LPS-activated RAW264.7 cells. Cells were incubated with tomato aqueous extract (TAE), secretion in LPS-activated RAW264.7 cells. Cells were incubated with tomato aqueous extract (TAE), stimulated with 1 μg/mL LPS and cultured for 24 h. “LPS only”: indicates the value obtained from stimulated with 1 µg/mL LPS and cultured for 24 h. “LPS only”: indicates the value obtained LPS-stimulated cells (without substance) and is indicated on the y-axis. Representative results from from LPS-stimulated cells (without substance) and is indicated on the y-axis. Representative results one of three independent series are shown as mean (±SD) of duplicate. * p < 0.05, ** p > 0.01 (vs. from one of three independent series are shown as mean (˘SD) of duplicate. * p < 0.05, ** p > 0.01 LPS-stimulated cells). Unstimulated cells produced 0.01 ± 0.00 μM NO and 133 ± 18 pg/mL PGE2. (vs. LPS-stimulated cells). Unstimulated cells produced 0.01 ˘ 0.00 µM NO and 133 ˘ 18 pg/mL PGE2 .
While TAE contained no detectable quantities of vitamin C, E and lycopene, it had significant WhileofTAE contained no detectable quantities vitamin C, E and lycopene, it to had amounts adenosine, chlorogenic acid (CA) and rutinof(Table 2), which could contribute thesignificant altered amounts of adenosine, acidanalyzed (CA) and (Table 2), which and could to the inflammatory response.chlorogenic Therefore, we therutin impact of adenosine thecontribute two phenolic compounds on RAW264.7 cells.Therefore, We observed that adenosine significantly modulated thetwo secretion altered inflammatory response. we analyzed the impact of adenosine and the phenolic of IL-6 and on TNF-α. CA andcells. rutin We blunted NO and IL-6, whereas significantly they had no substantial effect on the compounds RAW264.7 observed that adenosine modulated the secretion other mediators (Tableblunted 2). We also differences adenosine, CA andeffect rutin on ofsecretion IL-6 andofTNF-α. CA and rutin NOnoticed and IL-6, whereasbetween they had no substantial with regard to the regulation of gene expression: adenosine and TAE had similar effects on geneand the secretion of other mediators (Table 2). We also noticed differences between adenosine, CA regulation (Figure Materials Figure S1). Rutin and had a common pattern rutin with regard to 2, theSupplementary regulation of gene expression: adenosine andCA TAE had similar effects onon gene gene expression, which only partially overlapped with that of adenosine. regulation (Figure 2, Supplementary Materials Figure S1). Rutin and CA had a common pattern on
gene expression, which only partially overlapped with that of adenosine.
Table 2. Constituents of tomato aqueous extracts (TAE).
Table 2. Constituents of tomato aqueous extracts (TAE). 1 Constituent (In %) (μM)
Adenosine Constituent Rutin Adenosine Chlorogenic Acid Vitamin CRutin Chlorogenic Acid Vitamin E Vitamin C Lycopene
1.73 (In %) 1 0.592 1.73 0.182 2 0.592
